A challenge in gene therapy is the limited ability of vectors to transduce G0 resting CD4+ T cells and G0 stem cells. It is important to develop such vectors because transducing resting cells, as compared to activated cells, is more physiological, less expensive, less labor-intensive, possibly safer, and the gene-marked cells may be longer lasting in vivo. VSV-G protein is an envelope that is commonly used to pseudotype lentiviral vectors for transducing activated CD4+ T cells;however, lentiviral vectors pseudotyped with VSV-G do not transduce G0 CD4+ T cells efficiently. A long-term goal of our lab is to design a better envelope for transducing G0 resting CD4+ T cells. Therefore, we investigated why VSV-G pseudotypes do not transduce resting CD4+ T cells by measuring binding, fusion, reverse transcription, and integration in resting cells. Using this approach of assaying individual steps of HIV infection, we previously showed, contrary to dogma, that HIV transduces resting CD4+ T cells. When we compared lentiviral vectors pseudotyped with VSV-G or HIV envelope (Env), we found that HIV Env pseudotypes fused to resting CD4+ T cells 100 times more efficiently than VSV-G pseudotypes. Further investigation of this finding suggested that HIV Env fuses to resting cells much more efficiently because binding of HIV Env to co-receptor triggers signaling that induces cellular changes that enhance viral fusion. Toward the goal of designing a better envelope for transducing G0 resting CD4+ T cells, we will test the ability of other envelopes, singly and in combination with HIV Env, to fuse to resting T cells, and explore the mechanism underlying the ability of HIV Env to efficiently fuse to resting CD4+ T cells. PUBLIC HEALTH RELEVANCE: Gene therapy is developing into a viable alternative for the treatment of many diseases including immune system disorders, certain cancers, and even HIV infection. In gene therapy, a gene of interest is delivered to a target cell, which in some cases is a quiescent cell;however, to obtain efficient gene transfer it is often necessary to activate a quiescent cell to enter the cell cycle. An important but elusive goal is delivery of the gene of interest to certain quiescent cells without activating them, because cell cycling not only alters the function of the quiescent cell, but may also be a safety risk. In this grant we propose to identify methods that allow delivery of a gene to quiescent CD4+ T cells as the first step toward achieving the goal of gene delivery without cellular activation.